Combinatorial solid-phase synthesis and screening of a diversetripodal triazacyclophane (TAC)-based synthetic receptor library

showing a remarkable selectivity towards a D-Ala-D-Alacontaining ligand

Cristina Chamorro, Jan-Willem Hofman and Rob M. J. Liskamp*

Department of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, PO Box 80082,

3508 TB Utrecht, The Netherlands

Received 16 January 2004; accepted 4 May 2004

Abstract—A large and diverse library of a TAC-based tripodal synthetic receptor library (6) has been prepared by split-mix synthesis on thesolid phase. Each receptor of the 46,656-member TAC-based library (6) is attached to a solid support bead and contains three differentdipeptide arms. On-bead screening for binding of a D-Ala-D-Ala containing ligand (7) by the TAC-based library (6) was performed inphosphate buffer (0.2 N, pH¼7.0). Remarkable selectivity for particular library members was observed. The best binding members from thescreening were manually selected using fluorescence microscopy and subjected to structural analysis by sequencing. The thus determinedbinding sequences showed a high consensus.q 2004 Elsevier Ltd. All rights reserved.

1. Introduction

Design and synthesis of synthetic receptors molecules forselective recognition of bioactive molecules have receivedconsiderable attention in recent years.1 Synthetic receptorsfor specific peptide sequences may provide model systemsfor biologically relevant peptide-protein interactions andmay lead to applications in the area of biosensors,therapeutics and catalysis. Among the peptide containingreceptors, the well-studied tweezer-like synthetic receptorsthat contain two binding arms showed high selectivity forcertain peptide sequences.2,3 Since amino acid sequences ofthe receptor arms are crucial for the recognition of a ligand,one-bead-one-compound combinatorial solid phase syn-thesis,4 provides a powerful strategy for the generation of

libraries of synthetic receptors with attractive affinity and/orselectivity for specific peptide sequences.5 Preparation of alibrary on the solid phase using split-mix synthesis6 ensuresthat each single bead carries one particular receptor. As aresult, the library can be screened either manually or using abead sorter and the selected receptor beads can be subjectedto structural elucidation.

In order to increase diversity, possibly affinity andselectivity, we are interested in the development of tripodalsynthetic receptors that contain—compared to tweezers—an additional peptide-binding arm. Only a limited number oftripodal scaffolds suitable for attachment of three bindingarms have been described so far.7 We are particularlyinterested in tripodal triazacyclophane (TAC) and cyclo-triveratrylene (CTV) scaffolds and we have developedefficient syntheses for both.5a,8 In addition, their applica-bility for the preparation of tripodal receptors with two orthree identical peptidic arms has been described.5a,8

Receptors were uncovered with promising selectivity andbinding affinity towards binding of bacteria cell wallprecursors containing D-Ala-D-Ala or D-Ala-D-Lac5a,9

sequences. In order to significantly increase the structuraldiversity, a selectively deprotectable TAC scaffold (1)10

was developed. The presence of orthogonal protectinggroups such as fluorenylmethoxycarbonyl (Fmoc), 2-nitro-benzenesulfonyl (o-NBS), and allyloxycarbonyl (Aloc)allowed the selective introduction and elongation of three

0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.tet.2004.05.121

Tetrahedron 60 (2004) 8691–8697

* Corresponding author. Tel.: þ31-30-2537396; fax: þ31-30-2536655;e-mail address: r.m.j.liskamp@pharm.uu.nl

Keywords: Synthetic receptors; TAC-based library; Screening; Fluorescentligand; D-Ala-D-Ala-OH.

Abbreviations: Aloc, Allyloxycarbonyl; Boc, tert-butoxycarbonyl; BOP,benzotriazol-tris-(dimethylamino)phosphonium hexafluoro-phosphate;tBu, tert-butyl; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DCM,dichloromethane; DiPEA, N,N-diisopropyl-N-ethylamine; DMF,dimethylformamide; Et2O, diethyl ether; Fmoc, N-fluoren-9-ylmethoxy-carbonyl; HOBt, 1-hydroxybenzotriazol; o-NBS, 2-nitrobenzenesulfonyl;NMP, N-methylpyrrolidone; rt, room temperature; TAC, 1,5,9-triaza-3(1,3)-benzenacyclododecaphane-35-carboxylic acid; TEA, triethylamine;TFA, trifluoroacetic acid; TIS, triisopropylsilane.

different peptide arms.11 The suitability of 1 for theconstruction of TAC-based receptors was recently demon-strated by the preparation on the solid phase of diverseTAC-based libraries consisting of a considerable number oflibrary members having three different amino acids and/ordipeptides as binding arms (theoretically 225 and 27,000members, respectively).11

Here, we describe the construction of a diverse and largecombinatorial library of TAC-based tripodal syntheticreceptors on the solid phase. As an example for selectiverecognition of peptide sequences, the 46,656-member

TAC-based receptor library was screened for binding ofthe D-Ala-D-Ala sequence.

2. Results and discussion

2.1. Solid phase synthesis of the TAC-based library 6

A TAC-based tripodal receptor library (6) containing threedifferent peptide arms was prepared using the split-mixmethod.4,6 In order to facilitate screening experiments,the TAC-based receptors were covalently bound to

Scheme 1. Combinatorial solid phase synthesis of a large and diverse triazacyclophane (TAC)-based synthetic receptor library consisting of 46,656 members.

C. Chamorro et al. / Tetrahedron 60 (2004) 8691–86978692

Argogelw-NH2 resin. The strategy used for the preparationof library 6 is depicted in Scheme 1. The Argogelw-NH2

resin was loaded with TAC scaffold (1) and the threedifferent dipeptide arms (Arm 1, Arm 2, and Arm 3) wereassembled one by one onto the TAC scaffold in two cyclesafter subsequent cleavage of the corresponding orthogonalprotecting group. To ensure the possibility of completestructural elucidation by Edman degradation12 by havingunique amino acid sequences for each library member, threedifferent representative sets each containing six differentamino acids were chosen for the construction of the threepeptide arms (Scheme 1). The side chains of functionalamino acids were protected with acid-labile groups.Eighteen out of the twenty proteinogenic amino acidswere used excluding cysteine and methionine. Fmoc-protected amino acids were used as a building block forthe first cycle whereas Boc-protected amino acids were usedfor the second cycle in order to prevent further elongation.In the last, third arm, Fmoc-protected amino acids were used

for the second coupling cycle to determine the final averageloading of the library.

Thus, loading of Argogelw-NH2 resin using BOP13 as acoupling agent led to resin 2. After removal of the Fmocgroup, the resin was divided into six equal portions andBOP-coupling of the corresponding Fmoc-protected aminoacid of Set 1 [Fmoc-L-Lys(Boc)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Phe-OH, Fmoc-Gly-OH,Fmoc-L-Ser(tBu)-OH)] was performed. Negative chlora-nil14 and bromophenol blue (BPB)15 tests for the presence ofsecondary amines confirmed completion of the couplingreactions in all the cases. After mixing of the six portions,the Fmoc group was removed and the resin was split againinto 6 portions. After BOP-coupling of the second aminoacid, using the corresponding Boc-protected amino acids ofSet 1 [Boc-L-Lys(Boc)-OH, Boc-L-Glu(OtBu)-OH, Boc-L-Val-OH, Boc-L-Phe-OH, Boc-Gly-OH, Boc-L-Ser(tBu)-OH)], pooling was carried out to give library 3. Theo-NBS group was removed from the pooled resin bythiolysis16 and introduction and synthesis of the second arm(Arm 2) was performed analogously to the construction ofthe first arm. In this case, amino acid from Set 2 [Fmoc-L-His(Trt)-OH, Fmoc-L-Ala-OH, Fmoc-L-Leu-OH, Fmoc-L-Pro-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Asn(Trt)-OH] andtheir Boc-protected counterparts were used to afford 4. Afterpooling, the Aloc group was removed by Pd-catalyzed allyltransfer to anilinium p-toluenesulfinate10,17 and the thirdarm (Arm 3) was introduced similarly, using only Fmoc-protected amino acids from Set 3 [Fmoc-L-Arg(Pbf)-OH,Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ile-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gln(Trt)-OH], to give the fully protected TAC-based library 5.

Table 1. Sequences of the red beads

Bead Arm 1 Arm 3 Arm 2

AA2 AA1 AA6 AA5 AA4 AA3

1a Ser Gly Ile Thr His His2a Lys Ser Thr Gln His His3a Phe Gly Thr Gln His His4a Phe Gly Thr Thr His His5a Val Lys Thr Ile His His6b Val Glu Arg Arg His His7b Phe Lys Trp Arg His His

a Dark-red bead.b Light-red bead.

Figure 1. Screening results of the TAC-based library with a fluorescent D-Ala-D-Ala containing ligand and visualization of the found amino acids at eachposition in the peptide binding arms by a ‘pie’ representation.

C. Chamorro et al. / Tetrahedron 60 (2004) 8691–8697 8693

Finally, Fmoc-cleavage followed by global deprotection ofall functionalized side chains as well as Boc cleavage usingTFA/TIS/H2O was performed and the fully deprotectedTAC-based library 6 was obtained. The large and highlydiverse library, containing 46,656 different receptors (66),was now ready for screening of selective binding by specificpeptide sequences. Interestingly, a low percentage of red-colored beads were found. To determine the identity ofthose beads, five dark red beads together with two light-redbeads were selected and sequenced showing the presence ofHis-His sequence (AA3-AA4) in one of the outer arms (Arm2) (Table 1, beads 1–7). Apparently the red color wasassociated with the presence of His-His sequence in outerarm 2 (AA3, AA4). Since a Pd0-catalyst was used for Aloccleavage, a complex of Pd2þ and the His-His sequence,18

might have caused the red color. Only the dark-redbeads showed also the presence of Thr in the middle arm3 (AA5-AA6), indicating that the intensity of the red colormight depend on the amino acid composition of the middlearm (Table 1).

2.2. Screening for binding of D-Ala-D-Ala by the TAC-based library 6

As part of a program towards uncovering compoundscapable of binding to crucial structural elements ofpathogens, peptide sequence D-Ala-D-Ala present in thepeptidoglycan precursor needed for construction of the cellwall in growing gram-positive bacteria9 was chosen as aligand for assessing the binding properties of TAC-basedreceptor library 6. This might ultimately lead to syntheticreceptors capable of mimicking the binding and therebyantibiotic properties of vancomycin.19 In order to visualizethe best binding beads by screening, a ligand containing thefluorescent NBD label, that is, NBD-N(H)-(CH2)5-C(O)-Gly-D-Ala-D-Ala-OH (7)20 was used (Fig. 1).

Thus, screening for the ability to bind the D-Ala-D-Alacontaining ligand (7) by the TAC-based tripodal receptorlibrary 6 was performed in phosphate buffer (0.2 N,pH¼7.0) (Fig. 1) on ca 140,000 beads, roughly correspond-ing to three copies of each synthetic receptor. So far thereare not many examples of screening for binding by syntheticreceptors in aqueous systems.21 However, this was deemedabsolutely essential if one wants to move to biologicallyrelevant systems and find hits for these. We found that it isno longer difficult to find hits of synthetic receptors withgood to excellent binding properties in organic solvents22

The challenge, however, is to discover molecules with goodbinding properties in an aqueous environment, sinceintermolecular interactions (hydrogen bond, electrostatic)between ligand and receptor are much weaker here than inapolar solvents.

Upon incubation with fluorescent ligand 7 a high selectivityof binding was found as was judged by observation of arange of intensities of fluorescent beads under thefluorescence microscope (Fig. 1). The most intensive twelvefluorescent beads were selected and subjected to Edmandegradation.12 As a confirmation of the identity of thesynthetic receptors, Edman sequencing was carried out in allcases for an additional third cycle, which invariably showedthe absence of a third amino acid, thus confirming the length

of the peptide arms. As a negative control, a non fluorescentbead was selected and sequenced. The identity of thepeptide arms of the bead attached TAC-based receptors isshown in Table 2. Perusal of the data revealed a remarkableselectivity. First, completely different sequences wereobtained for the negative control as compared to thefluorescent beads. Interestingly, identical or very similaramino acid sequences were found in the receptors present onthe selected fluorescent beads. Despite the fact that histidinewas found at position AA3 or AA4 in some of the fluorescentbeads, none of them were colored red (vide supra) indicatingthat the His-His sequence was required for this property(Table 1). The frequency of the found amino acids at eachposition is shown with a ‘pie’ representation of each aminoacid residue (Fig. 1). From Table 2 the following trends canbe inferred. Basic amino acids (blue) were especially foundat the end of the arms (AA2: Lys 67%, AA4: His 25%, AA6:Arg 75%) and predominantly in arms 1 (Lys) and 3 (Arg).Arg was also found at AA5(33%) in arm 3. Hydrophobicamino acids (green) were often found closest to the scaffold(AA1: Phe 83%, AA3: Leu/Pro 50% and Tyr 50%, AA5: Ile58%). However, AA4 had usually a fairly hydrophobiccharacter, and Tyr, Ala and Leu accounted for 67% of theamino acids in the sequenced beads at his position.Combination of a basic amino acid and a hydrophobicamino acid was especially prominent in arms 1 and 3.

Remarkable is the number of times an identical sequencewas found in arm 1 or arm 3. Lys-Phe was found in arm 1 in8 out of 12 sequenced beads, that is, 66%. Arg-Ile in arm 3was found in 6 out of 12 beads, that is, 50%. Thecombination of the these two sequences, that is, Lys-Phein arm 1 and Arg-Ile in arm 3 was even found in 4 out of 12beads, that is, in 33% of the sequenced beads.

These data point to remarkable selectivity of binding, sincethis combination sequence is only present in 36 (62: possiblecombinations of amino acids in arm 2) library members inthe total library of 46,656, corresponding to only 0.08% ofthe beads.

Based on these data it is tempting to speculate about apossible binding mode of ligand 7 to a ‘cavity’ formed byarm 1 and arm 3 in the sequenced synthetic receptors.

Table 2. Sequences of synthetic receptors for 7

Bead Arm 1 Arm 3 Arm 2

AA2 AA1 AA6 AA5 AA4 AA3

8a Lys Phe Arg Ile His Tyr9a Lys Phe Arg Ile Tyr Tyr10a Lys Phe Arg Ile His Pro11a Lys Phe Arg Ile Ala Leu12a Glu Phe Arg Ile Tyr Pro13a Glu Lys Arg Ile Tyr Leu14a Lys Phe Arg Arg Tyr Leu15a Lys Phe Arg Arg Leu Tyr16a Lys Phe Ile Arg Leu Tyr17a Ser Glu Ile Arg His Pro18a Lys Phe Ile Ile Tyr Tyr19a Glu Phe Arg Gln Asn Tyr20b Phe Phe Asp Gln Asn His

a Fluorescent bead.b Non-fluorescent, control bead.

C. Chamorro et al. / Tetrahedron 60 (2004) 8691–86978694

Hydrophobic interactions or aromatic p-stacking might bepossible between the fluorescent label of the ligand (NBDgroup) and the branched or aromatic side chains ofpredominantly Phe (AA1) in arm 1 or Tyr (AA3) in arm 2and Ile (AA5) in arm 3, which are located close to the TAC-scaffold. Ionic interactions are likely between the Ala-carboxylate terminus of ligand 7 and the amino termini and/or the basic side chains of outer amino acids Lys (AA2) inArm 1 and Arg (AA6) in Arm 3. Although Arm 2 seemed inthis model to be less involved in the binding, which was alsoperceptible from the higher variability of amino acids in thisarm, hydrogen bond formation with the backbones amidebonds and/or hydrophobic interactions with the Ala-Megroups are possible. As a consequence the role ofhydrophobic amino acids might also be to create ahydrophobic environment in the aqueous medium ofscreening, which is favorable for binding. Such a situationis distantly related to the hydrophic cavities created by manyenzymes.23

3. Conclusions

A convenient split-mix protocol was used for the prepa-ration of a large and diverse synthetic receptor library basedon the TAC-scaffold, which allowed the introduction ofthree different (peptide) arms. Although of considerable size(46,656 members), the size of the library can be easilyincreased by the introduction of longer arms and armscontaining up to ten amino acids have been introduced.Screening for binding of the fluorescent D-Ala-D-Alacontaining ligand (7) by the tripodal receptor library 6was carried out in phosphate buffer aqueous system and ledto the identification of selective synthetic receptors.

Under present investigation is the preparation of largerlibraries and screening with other ligands.

4. Experimental

4.1. General

All reagents were purchased from commercial sources andused without further purification. Argogelw-NH2

(0.40 mmol/g, average bead diameter 178 mm) resin waspurchased from Argonaut Technologies, Inc. Protectedamino acids were purchased from Alexis Corporations(Laufelfingen, Switzerland) and Advanced ChemtechEurope (UK). All reactions on the solid phase wereperformed in standard glassware or poly(ethylene) glycol(PE) syringes with PE frits. Peptide grade solvents, dried onmolecular sieves were used for reactions and resin washingsteps. The used capping reagent was a mixture of aceticanhydride (42 mL), DiPEA (2.18 mL), HOBT (0.23 g), andNMP (100 mL). Anilinium p-toluenesulfinate24 wasobtained by reaction of p-toluenesulfinic acid sodium saltwith aniline in DCM and crystallized upon slow adition ofhexane. DiPEA was subsequently distilled from KOH andninhydrin. For Fmoc determinations, a Perkin ElmerLambda 2 UV/VIS spectrometer was used. Kaiser,25

bromophenol blue (BPB),15 and chloranil tests14 wereused for detection of remaining primary and/or secondary

amines on the solid phase. Edman degradations12 wereperformed on an Applied Biosystems ABI 476A proteinsequencer. Polymer beads were visualized and manipulatedunder a Leica MZ FL III microscope equiped with a CCDcamera.

4.2. General procedure for coupling Fmoc/Boc-aminoacids on the solid phase

The resin (1 equiv.) was swollen in NMP (2 min) and, afterdraining the solvent, BOP (4 equiv.), Fmoc/Boc-amino acid(4 equiv.) and NMP (15 mL/mmol) were added. Themixture was shaken until complete disolution and DiPEA(8 equiv.) was added. After shaking for 4 h, the resin waswashed with NMP (5£7 mL, each 2 min) and DCM(5£8 mL, each 2 min). Negative Kaiser, BPB tests and/orchloranil test indicated completion of the coupling reaction.After drying the resin in vacuo overnight, loading of theresin was assessed by Fmoc-determination.

4.3. General procedure for Na-Fmoc deprotection

Na-Fmoc-protected resin was swollen in NMP (2 min) and,after draining the solvent, the resin was shaken with 20%piperidine in NMP (3£10 mL/mmol, each 10 min). Theresin was washed with NMP (5£2 min) and DCM(5£2 min). Positive Kaiser, BPB and/or chloranil testsindicated the successful Fmoc-deprotection.

4.4. Solid phase synthesis of library 6

Argogelw-NH2 (6.25 g, 2.5 mmol) was swollen in NMP(38 mL, 2 min). After draining the solvent, 1 (1.92 g,2.5 mmol), BOP (1.10 g, 2.5 mmol) and NMP (38 mL) wereadded and a gentle stream of dry nitrogen was bubbledthough the mixture until all reagents were dissolved. DiPEA(0.87 mL, 5 mmol) was added and N2 bubbling wascontinued overnight. The resin was drained and washedwith NMP (5£38 mL, each 2 min), DCM (5£38 mL, each2 min) and Et2O (5£38 mL, each 2 min). After drying invacuo overnight, the loading of the resin was determined(0.33 mmol g21). Remaining free amines on the resin wereacetylated by addition of the capping agent (15 mL/mmol)and shaking for 1 h. After draining, the resin was washedwith NMP (5£38 mL, each 2 min) and DCM (5£38 mL,each 2 min). After Na-Fmoc deprotection, resin 2 was driedunder vacuo overnight and divided into six equal portions(1.13 g, 0.33 mmol) in PE syringes with PE frits. each resinportion was swollen in NMP (5 mL, 2 min) and afterdraining the solvent, coupling of a different Fmoc-aminoacid from Set 1 for AA1 [Fmoc-L-Lys(Boc)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Phe-OH, Fmoc-Gly-OH, Fmoc-L-Ser(tBu)-OH)] in each syringe was carriedout following the general procedure. The average loadingwas calculated from each individual loading and was0.32 mmol.g21. The content of the syringes was pooledand mixed in a reaction vessel, washed with NMP(2£30 mL, each 2 min) and Na-Fmoc was cleaved follow-ing the general protocol. Then, the split-mix procedure wasrepeated for the coupling of the second amino acid. Thecorresponding Boc-amino acid from Set 1 for AA2 [Boc-L-Lys-(Boc)-OH, Boc-L-Glu(OtBu)-OH, Boc-L-Val-OH,Boc-L-Phe-OH, Boc-Gly-OH, Boc-L-Ser(tBu)-OH)] were

C. Chamorro et al. / Tetrahedron 60 (2004) 8691–8697 8695

used to afford library 3. Upon drying in vacuo overnight, 3(2.08 mmol) was washed with DMF (5£30 mL, each 2 min)and dry (molecular sieves) DMF (5£30 mL, each 2 min).The solvent was drained again and the o-NBS group wascleaved by addition of DMF (30 mL), DBU (1.57 mL,10.40 mmol, 5 equiv.) and b-mercaptoethanol (1.46 mL,20.80 mmol, 10 equiv). After N2 bubbling for 30 min, thegreen solution was replaced by a fresh mixture of identicalcomposition and N2 bubbling was maintained for another30 min. The resin was washed with NMP (5£30 mL, each2 min), DCM (5£30 mL, each 2 min) and Et2O (5£30 mL,each 2 min) and dried under vacuo overnight. The split-mixprocedure described above for the construction of the firstarm was repeated for the second arm, using amino acidsfrom Set 2 [Fmoc-L-His(Trt)-OH, Fmoc-L-Ala-OH, Fmoc-L-Leu-OH, Fmoc-L-Pro-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Asn(Trt)-OH] for AA3 or for AA4 their Boc-protectedanalogues to give library 4. lastly, the Aloc group wascleaved. After swelling of library 4 (2.0 mmol) in NMP(30 mL, 2 min) and draining the solvent, anilinium p-toluensulfinate (9.92 g, 40 mmol, 20 equiv) and NMP(30 mL) were added. A gentle stream of dry nitrogen wasbubbled though the mixture for 5 min and tetrakis(triphe-nylphosphine)-palladium(0) (0.35 g, 0.3 mmol, 15 mol%)was added. N2 bubbling was maintained for 1 h underexclusion of light. The resin was washed with NMP(3£30 mL, each 2 min), 0.1% solution of sodium diethyl-dithiocarbamate trihydrate in NMP (1£30 mL, each 2 min),a 20% solution of DiPEA in NMP (1£30 mL, each 2 min),NMP (5£30 mL, each 2 min), DCM (4£30 mL, each 2 min)and Et2O (5£30 mL, each 2 min) and dried under vacuoovernight. For the construction of the third arm, only Fmoc-protected amino acid from Set 3 for both AA5 and AA6

[Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ile-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Thr(tBu)-OH,Fmoc-L-Gln(Trt)-OH] were used to furnish library 5 havingan average loading of receptors equivalent to0.32 mmol g21 as was assessed by Fmoc-determination.After final pooling, the resin was washed with NMP(5£30 mL, each 2 min), DCM (5£30 mL, each 2 min) andEt2O (5£30 mL, each 2 min) and stored in vacuo.

A small portion of the library 5 (0.25 g, 0.08 mmol) wassubjected to global deprotection by Fmoc-deprotection,followed by treatment of the resin with a mixture of TFA:H2O: TIS, 95:2.5:2.5 (v/v/v) (5 mL) for 4 h. The resultingresin was washed with NMP (3£1.2 mL, each 2 min), aceticacid (0.1 M) (3£1.2 mL, each 30 min), NMP (5£1.2 mL,each 2 min), 25% DiPEA in NMP (5£1.2 mL, each 2 min),NMP (5£1.2 mL, each 2 min), DCM (5£2 mL, each 2 min),dioxane (4£1.2 mL, each 2 min), dioxane: H2O, (1:1)(4£1.2 mL, each 2 min), H2O (4£1.2 mL, each 2 min),and Et2O (4£1.2 mL, each 2 min), to give the fullydeprotected library 6.

4.5. Screening for binding of NBD-N(H)-(CH2)5-C(O)-Gly-D-Ala-D-Ala-OH by the TAC-based library

TAC-based synthetic receptor library 6 (0.18 g,0.079 mmol, ,140.000 beads, ,3 copies/receptor) wassuspended in a 46 mM solution of NBD-N(H)-(CH2)5-C(O)-Gly-D-Ala-D-Ala-OH (7) in phosphate buffer (0.2 N,pH¼7.0) (158 mL) and incubated with gentle shaking and

exclusion of light at 20 8C for 65 h. Then resin was drainedand washed with phosphate buffer (0.2 N, pH¼7.0)(5£158 mL, each 2 min). Then, resin was poured into apetri dish and spread into a monolayer. The beads wereviewed under a fluorescence microscope. By use of a longneedle, most fluorescent or colored beads were isolated(,180 beads). The fluorescence of these preselected beadswas reevaluated using the overexposure mode of the LeicaDC-100 digital camera system and image analysis toestimate the relative fluorescence intensities semi-quanti-tatively. The best twelve fluorescent beads were selectedand analysed by parallel Edman degradation together with anon-fluorescent bead and four of the found red beads.

Acknowledgements

The Sequence Centre Utrecht is acknowledged for carryingout the sequence analyses. This work was supported by theEuropean Commission (Marie Curie Individual Fellowship,contract No. HPMFCT-2000-00704).

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